Targeted recombination of exogenous DNA to its homologous chromosomal location is a potentially powerful tool for correcting genetic defects in humans. Targeted recombination is well-documented in mammalian cells but it occurs at low frequency and is usually masked by a 100 to 10000 fold higher frequency of random integration. At present we know very little about either mechanism for incorporating exogenous DNA into chromosomes; nor do we understand the experimental or biological parameters that influence these two processes. As a result, it is difficult to design targeting vectors and experimental procedures that maximize homologous recombination and minimize random integration. The goal of this project is to design and test appropriate vectors for the correlation of genetic defects. We are especially interested in the correction of genetic deletions. Deletions are a very common kind of defect in patients and they present novel theoretical and practical challenges that must be overcome if such patients are to be helped by targeted recombination. We also feed that studying the correction of deletions will yield substantial basic information about the mechanism of targeted recombination. Our proposed studies will exploit the unique properties of the human HPRT gene, including forward and reverse selection, defined deletions, and a known sequence. These features will allow us to test efficiently a variety of targeting parameters, including the total length of homology, the distribution of homology around the deletion, the size of the deletion, the position of the deletion within the gene, and the influence of specific sequences surrounding the deletion. In addition, we intend to develop protocols for repairing deletions that are too large to correct to the wild type sequence. Our approach will be to substitute missing exons by laying in a "patch" of cDNA. We anticipate that our results will yield a set of basic design principles that will allow targeted correction of unselectable loci.